Biological Evaluation of Triorganotin Derivatives as Potential Anticancer Agents

Metal-derived platinum complexes are widely used to treat solid tumors. However, systemic toxicity and tumor resistance to these drugs encourage further research into similarly effective compounds. Among others, organotin compounds have been shown to inhibit cell growth and induce cell death and autophagy. Nevertheless, the impact of the ligand structure and mechanisms involved in the toxicity of organotin compounds have not been clarified. In the present study, the biological activities of commercially available bis(tributyltin) oxide and tributyltin chloride, in comparison to those of specially synthesized tributyltin trifluoroacetate (TBT-OCOCF3) and of cisplatin, were assessed using cells with different levels of tumorigenicity. The results show that tributyltins were more cytotoxic than cisplatin in all the tested cell lines. NMR revealed that this was not related to the interaction with DNA but to the inhibition of glucose uptake into the cells. Moreover, highly tumorigenic cells were less susceptible than nontumorigenic cells to the nonunique pattern of death induced by TBT-OCOCF3. Nevertheless, tumorigenic cells became sensitive when cotreated with wortmannin and TBT-OCOCF3, although no concomitant induction of autophagy by the compound was detected. Thus, TBT-OCOCF3 might be the prototype of a family of potential anticancer agents.


Introduction
The success of cis-diaminedichloroplatinum (II) (cisplatin) and of the second-generation derivatives carboplatin and oxaliplatin has opened a new perspective for the development of metal-based drugs for the treatment of several solid tumors [1]. Although this class of chemotherapeutic agents has a broad anticancer spectrum and is also used in combination, their systemic toxicity and tumor drug resistance have encouraged ongoing research for new metal-based drugs. Over the years, organotin compounds (i.e., nonplatinum but Table 1. 1 H and 13 C chemical shifts of the three tin compounds measured in phosphate buffer (pH 7.4) and DMSO-d 6 at 37 • C using TSP as a reference.

Position 1 Buffer DMSO
as the main mechanism possibly involved in the potential anticancer effect of organotin compounds [24][25][26], the range of molecular targets presumably involved is highly heterogeneous and awaits further studies and clarifications. Moreover, given the multiple potential activities of organotin derivatives towards cancer cells, the relationships among structures, physiochemical properties, and biological activities of this class of compounds, and whether a difference exists in the effect they exert on nontumorigenic cells compared with tumorigenic cells are still elusive.
In the present study, we explored the physiochemical properties of TBT-O and TBT-Cl derivatives in a hydrophilic environment and their dose-dependent cytotoxic effects towards representative high-tumorigenic and nontumorigenic cells in comparison with a tributyltin trifluoroacetate (TBT-OCOCF3). This organotin compound was specially designed and synthetized to find a possible balance between toxicity and feasible antitumor activity. The possible mechanisms involved in the effects exerted by the compound on cell death were also investigated. Finally, based on the obtained results, a strategy of a combination treatment specifically aimed at enhancing the potential anticancer activity of similar organotin compounds is proposed.

NMR Characterization of Structure, Solubility, and Aggregation
The structures of the three investigated TBT compounds, TBT-O, TBT-Cl, and TBT-OCOCF3, were confirmed by NMR spectroscopy using 1 H-NMR, 1 H-1 H COSY, and 1 H- 13 C HSQC experiments (Figures S1). The chemical shifts obtained in DMSO-d6 and phosphate buffer at pH 7.4 are listed in Table 1. The chemical shift observed for the hydrogens in all compounds shows an accentuated shielding effect by Sn, which causes the signal of the CH2 group directly attached to it to occur at higher fields than those of the other two CH2 groups. The effect extends both to the hydrogens and the carbon of the CH2-1 group. This particular effect has already been observed in other cases [27].
The solubility was determined by preparing a 600 µL solution for each compound in the DMSO and buffer solution with a nominal concentration of 400 µM. The effective concentrations were measured at 37 °C using 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid (TSP) as an internal standard. The experimental and nominal values were almost identical for the DMSO solutions, whereas the measured values were significantly lower in the buffer solution. The three compounds showed solubility in the range of 40-70 µM in phosphate buffer, except for TBT-O, which was slightly less soluble ( Table 2). as the main mechanism possibly involved in the potential anticancer effect of organotin compounds [24][25][26], the range of molecular targets presumably involved is highly heterogeneous and awaits further studies and clarifications. Moreover, given the multiple potential activities of organotin derivatives towards cancer cells, the relationships among structures, physiochemical properties, and biological activities of this class of compounds, and whether a difference exists in the effect they exert on nontumorigenic cells compared with tumorigenic cells are still elusive.
In the present study, we explored the physiochemical properties of TBT-O and TBT-Cl derivatives in a hydrophilic environment and their dose-dependent cytotoxic effects towards representative high-tumorigenic and nontumorigenic cells in comparison with a tributyltin trifluoroacetate (TBT-OCOCF3). This organotin compound was specially designed and synthetized to find a possible balance between toxicity and feasible antitumor activity. The possible mechanisms involved in the effects exerted by the compound on cell death were also investigated. Finally, based on the obtained results, a strategy of a combination treatment specifically aimed at enhancing the potential anticancer activity of similar organotin compounds is proposed.

NMR Characterization of Structure, Solubility, and Aggregation
The structures of the three investigated TBT compounds, TBT-O, TBT-Cl, and TBT-OCOCF3, were confirmed by NMR spectroscopy using 1 H-NMR, 1 H-1 H COSY, and 1 H- 13 C HSQC experiments (Figures S1). The chemical shifts obtained in DMSO-d6 and phosphate buffer at pH 7.4 are listed in Table 1. The chemical shift observed for the hydrogens in all compounds shows an accentuated shielding effect by Sn, which causes the signal of the CH2 group directly attached to it to occur at higher fields than those of the other two CH2 groups. The effect extends both to the hydrogens and the carbon of the CH2-1 group. This particular effect has already been observed in other cases [27].
The solubility was determined by preparing a 600 µL solution for each compound in the DMSO and buffer solution with a nominal concentration of 400 µM. The effective concentrations were measured at 37 °C using 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid (TSP) as an internal standard. The experimental and nominal values were almost identical for the DMSO solutions, whereas the measured values were significantly lower in the buffer solution. The three compounds showed solubility in the range of 40-70 µM in phosphate buffer, except for TBT-O, which was slightly less soluble (Table 2). Thus, despite most authors focusing on the induction of the apoptotic form of RCD as the main mechanism possibly involved in the potential anticancer effect of organotin compounds [24][25][26], the range of molecular targets presumably involved is highly heterogeneous and awaits further studies and clarifications. Moreover, given the multiple potential activities of organotin derivatives towards cancer cells, the relationships among structures, physiochemical properties, and biological activities of this class of compounds, and whether a difference exists in the effect they exert on nontumorigenic cells compared with tumorigenic cells are still elusive.
In the present study, we explored the physiochemical properties of TBT-O and TBT-Cl derivatives in a hydrophilic environment and their dose-dependent cytotoxic effects towards representative high-tumorigenic and nontumorigenic cells in comparison with a tributyltin trifluoroacetate (TBT-OCOCF3). This organotin compound was specially designed and synthetized to find a possible balance between toxicity and feasible antitumor activity. The possible mechanisms involved in the effects exerted by the compound on cell death were also investigated. Finally, based on the obtained results, a strategy of a combination treatment specifically aimed at enhancing the potential anticancer activity of similar organotin compounds is proposed.

NMR Characterization of Structure, Solubility, and Aggregation
The structures of the three investigated TBT compounds, TBT-O, TBT-Cl, and TBT-OCOCF3, were confirmed by NMR spectroscopy using 1 H-NMR, 1 H-1 H COSY, and 1 H-13 C HSQC experiments (Figures S1). The chemical shifts obtained in DMSO-d6 and phosphate buffer at pH 7.4 are listed in Table 1. The chemical shift observed for the hydrogens in all compounds shows an accentuated shielding effect by Sn, which causes the signal of the CH2 group directly attached to it to occur at higher fields than those of the other two CH2 groups. The effect extends both to the hydrogens and the carbon of the CH2-1 group. This particular effect has already been observed in other cases [27].
The solubility was determined by preparing a 600 µL solution for each compound in the DMSO and buffer solution with a nominal concentration of 400 µM. The effective concentrations were measured at 37 °C using 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid (TSP) as an internal standard. The experimental and nominal values were almost identical for the DMSO solutions, whereas the measured values were significantly lower in the buffer solution. The three compounds showed solubility in the range of 40-70 µM in phosphate buffer, except for TBT-O, which was slightly less soluble (Table 2). The solubility was determined by preparing a 600 µL solution for each compound in the DMSO and buffer solution with a nominal concentration of 400 µM. The effective concentrations were measured at 37 • C using 3-(trimethylsilyl) propionic 2,2,3,3-d 4 acid (TSP) as an internal standard. The experimental and nominal values were almost identical for the DMSO solutions, whereas the measured values were significantly lower in the buffer solution. The three compounds showed solubility in the range of 40-70 µM in phosphate buffer, except for TBT-O, which was slightly less soluble (Table 2). The aggregation state of the three compounds in the aqueous buffer was determined by measuring their hydrodynamic radii (R h ) using 1,4-dioxane as an internal standard [28]. The hydrodynamic radius of each compound (R h,comp ) can be calculated using Equation (1): where R h,dioxane is the hydrodynamic radius of dioxane (2.12 Å). The diffusion coefficients of dioxane (D dioxane ) and the compound (D comp ) were measured using pulse-field gradient experiments. The results in DMSO show that all three compounds have a similar molecular volume (Table 2), even though a higher value of R h should be expected for TBT-O. Based on the R h value, we can conclude that, in DMSO, it is present as a monomer of its hydrated form, i.e., tributyltin hydroxide (TBT-OH), as shown in Table 1.
When compared with those obtained in DMSO, the R h values in phosphate buffer were higher for all compounds, except TBT-OCOCF 3 ( Table 2). This latter result can be explained by a hydrophobic collapse event caused by water, but it also indicates that this compound does not aggregate in the buffer solution. On the other hand, TBT-Cl shows the highest R h value in DMSO, implying that this compound is highly aggregated at its maximum concentration in the buffer solution. We performed a series of 1 H-NMR spectra experiments, lowering the TBT-Cl concentration to 25 µM without noticing a difference in the line shape (data not shown). This observation indicates that this compound also remains aggregated at lower concentrations.
TBT-O also shows an increase in its R h from DMSO to phosphate buffer. Two effects can contribute to increasing this value: presence of the oxide or formation of aggregates. We noticed a chemical shift difference between DMSO and phosphate buffer for hydrogens in positions 1 and 2, which might be compatible with the presence of the oxide TBT-O (Table 1).
However, we also observed a significant broadening for all signals in the water environment ( Figure 1), which are larger than expected for converting the monomer into the dimer. For this reason and for the significant increment in R h , we concluded that TBT-O is aggregated in phosphate buffer at 42 µM.
Finally, all compounds were stable in DMSO and phosphate buffer for several days, as judged from the invariance of the corresponding 1 H-NMR spectra (data not shown).  Finally, all compounds were stable in DMSO and phosphate buffer for as judged from the invariance of the corresponding 1 H-NMR spectra (data n

Effect of Organotin Compounds on the Metabolic Activity of CAL27, MCF-10 Cells, and PBMCs
Firstly, an MTT assay was performed as a biological indicator of the cellu activity, proliferation, and cytotoxicity in high-tumorigenic CAL27 cells and igenic MCF-10A cells exposed to the three organotin compounds under (TBT-O, TBT-Cl, and TBT-OCOCF3) and to the metal-derived anticancer d as a reference compound. In parallel, peripheral blood mononuclear cell healthy individuals and U937 cells were used as cellular models suitable for investigation. Five thousand cells were treated with the compounds at c ranging from 0.625 µM to 80 µM, and, after 24 h, the formazan production The results, expressed as half-maximal inhibitory concentration, are shown garding the high-tumorigenic CAL-27 cells, the results show that TBT-OCO an IC50 of 2.45 ± 0.14, i.e., lower than TBT-O (13.18 ± 3.70) but higher than T 0.53). On the other side, CAL-27 cells were much more resistant to cisplatin (1 than all of the tested organotins. Regarding the nontumorigenic MCF-10A and U937 cells, Table 3 shows that, even in these cells, TBT-OCOCF3 is m than cisplatin. TBT-O showed a metabolic inhibitory activity similar to OCOCF3 in PBMCs and U937 cells, respectively, while it exerted less inhib against MCF-10A cells (4.93 ± 2.33). Conversely, TBT-Cl is the most potent in pound showing an IC50 of approximately or lower than 1 µM in all tested ce Table 3. Inhibition of the metabolic activity assessed by the MTT assay in CAL-27 cells, PBMCs, and U937 cells treated with the organotin derivatives or cisplatin for 2  Firstly, an MTT assay was performed as a biological indicator of the cellular metabolic activity, proliferation, and cytotoxicity in high-tumorigenic CAL27 cells and in nontumorigenic MCF-10A cells exposed to the three organotin compounds under investigation (TBT-O, TBT-Cl, and TBT-OCOCF 3 ) and to the metal-derived anticancer drug, cisplatin, as a reference compound. In parallel, peripheral blood mononuclear cells (PBMCs) of healthy individuals and U937 cells were used as cellular models suitable for a cytotoxicity investigation. Five thousand cells were treated with the compounds at concentrations ranging from 0.625 µM to 80 µM, and, after 24 h, the formazan production was assessed. The results, expressed as half-maximal inhibitory concentration, are shown in Table 3. Regarding the high-tumorigenic CAL-27 cells, the results show that TBT-OCOCF 3 exhibited an IC 50 of 2.45 ± 0.14, i.e., lower than TBT-O (13.18 ± 3.70) but higher than TBT-Cl (0.91 ± 0.53). On the other side, CAL-27 cells were much more resistant to cisplatin (138.60 ± 59.52) than all of the tested organotins. Regarding the nontumorigenic MCF-10A cells, PBMCs, and U937 cells, Table 3 shows that, even in these cells, TBT-OCOCF 3 is more inhibitory than cisplatin. TBT-O showed a metabolic inhibitory activity similar to that of TBT-OCOCF 3 in PBMCs and U937 cells, respectively, while it exerted less inhibitory activity against MCF-10A cells (4.93 ± 2.33). Conversely, TBT-Cl is the most potent inhibitory compound showing an IC 50 of approximately or lower than 1 µM in all tested cells.

Effects of Triorganotin Compounds on Cell Viabilities of CAL-27 and MCF-10A Cells
The viabilities of the CAL-27 and MCF-10A cells were determined by the trypan blue exclusion test after the treatment of these cells (1 × 10 5 ) with TBT-O, TBT-Cl, and TBT-OCOCF 3   The addition of TBT-OCOCF3 more slightly affected the decrease in MCF-10A viable cells, with an inhibition of viability by 90% only at 20 µM ( Figure 2b). Taken together, these data suggest a milder cytotoxic activity of TBT-OCOCF3 in comparison with the other triorganotin derivatives against the low-tumorigenic cells. Interestingly, the CAL-27 and MCF-10A cells were resistant to cisplatin treatment even at the higher concentration tested (Figures 2a,b). When the same assay was performed with the less cytotoxic TBT-OCOCF3 organotin compound in nonadherent U937 cells, after 24 h of treatment at The addition of TBT-OCOCF 3 more slightly affected the decrease in MCF-10A viable cells, with an inhibition of viability by 90% only at 20 µM (Figure 2b). Taken together, these data suggest a milder cytotoxic activity of TBT-OCOCF 3 in comparison with the other triorganotin derivatives against the low-tumorigenic cells. Interestingly, the CAL-27 and MCF-10A cells were resistant to cisplatin treatment even at the higher concentration tested (Figure 2a,b). When the same assay was performed with the less cytotoxic TBT-OCOCF 3 organotin compound in nonadherent U937 cells, after 24 h of treatment at 10 µM, only less than 10% of the viable cells in comparison with the control samples was still detectable ( Figure S2 in the Supplementary Materials). This result is coherent with that observed for the MTT assays in the same cells (Table 3).
The overall results of these experiments show an evident and, in some cases, dramatic dose-dependent disappearance of trypan-blue-negative cells showing plasma membrane integrity after TBT treatment. Surprisingly, however, this disappearance was not mirrored by a corresponding increase in the number of trypan-blue-positive dead cells. The presence of still morphologically intact, trypan-blue-positive cells was detectable, even if in small amounts, only in samples treated with the higher concentrations tested (10 µM and 20 µM), where, in any case, the sum of trypan blue-negative plus trypan blue-positive cells was never equivalent to the absolute number of control cells (data not shown).

Interaction of TBT-OCOCF 3 with 12 bp-DNA
In order to investigate the mechanisms underlying the cytotoxicity exerted by TBT-OCOF 3 , a first approach was to verify whether they were based on a direct interaction with DNA, similar to what was described for metal derivatives such as cisplatin. To this purpose, we investigated if TBT-OCOCF 3 could bind to DNA using NMR. For TBT-OCOCF 3 , the 1D and 2D spectra of 12 bp DNA were acquired in the presence or absence of the compound. As a positive control for the interaction, cisplatin was assayed, which forms adducts or inter-and intra-strand crosslinks resulting in interference in the replication machinery, G2/M cell arrest, and cell death by apoptosis or necrosis [29]. A solution containing 0.2 mM of the duplex, whose sequences are indicated in Figure 3, was annealed directly in the NMR tube. The double-helix formation was confirmed by the appearance of low-field signals belonging to the T-and G-NH groups (Figure 3a

Interaction of TBT-OCOCF3 with 12 bp-DNA
In order to investigate the mechanisms underlying the cytotoxicity exerted by TBT-OCOF3, a first approach was to verify whether they were based on a direct interaction with DNA, similar to what was described for metal derivatives such as cisplatin. To this purpose, we investigated if TBT-OCOCF3 could bind to DNA using NMR. For TBT-OCOCF3, the 1D and 2D spectra of 12 bp DNA were acquired in the presence or absence of the compound. As a positive control for the interaction, cisplatin was assayed, which forms adducts or inter-and intra-strand crosslinks resulting in interference in the replication machinery, G2/M cell arrest, and cell death by apoptosis or necrosis [29]. A solution containing 0.2 mM of the duplex, whose sequences are indicated in Figure 3, was annealed directly in the NMR tube. The double-helix formation was confirmed by the appearance of low-field signals belonging to the T-and G-NH groups (Figure 3a  After incubation with two equivalents of cisplatin for six hours, the NMR spectrum showed evidence of the interaction (Figure 3b). On the contrary, incubation with two or five equivalents of TBT-OCOCF3 did not result in any observable change in the NMR spectrum (Figure 3c). The signals of the compound were clearly observable in the mixture, indicating that it was available in solution for the interaction. These results indicate that TBT-OCOCF3 does not interact with the 12 bp DNA, suggesting that it is very likely that, differently from cisplatin, a direct interaction with DNA is not the mechanism of action of the cytotoxicity exerted by this compound or possibly other compounds of this class. After incubation with two equivalents of cisplatin for six hours, the NMR spectrum showed evidence of the interaction (Figure 3b). On the contrary, incubation with two or five equivalents of TBT-OCOCF 3 did not result in any observable change in the NMR spectrum ( Figure 3c). The signals of the compound were clearly observable in the mixture, indicating that it was available in solution for the interaction. These results indicate that TBT-OCOCF 3 does not interact with the 12 bp DNA, suggesting that it is very likely that, differently from cisplatin, a direct interaction with DNA is not the mechanism of action of the cytotoxicity exerted by this compound or possibly other compounds of this class.

Inhibition of Glucose Uptake by Triorganotins
Glucose metabolism plays a key role in tumor cell growth. Therefore, it was investigated whether the cytotoxicity exerted by the triorganotin compounds could be related to the inhibition of glucose uptake in the tumor or in the nontumorigenic cell lines. CAL-27 and MCF-10A cells were treated with TBT-O and TBT-OCOCF 3   Collectively, these data show that the organotin-derived compounds induce an immediate glucose uptake inhibition related to their cytotoxicity.

Effects of TBT-OCOCF3 on Cell Death in CAL-27 and MCF-10A Cells
Data on the inhibition of the metabolic activity and the viable/dead cell count indicate that the CAL-27 cells were resistant to the cytotoxic effects exerted by cisplatin but quite susceptible to those induced by organotin derivatives, including TBT-OCOCF3. However, except for a certain relationship between toxicity and glucose uptake inhibition, the mechanisms involved in the cytotoxic effects of the organotin derivatives, particularly TBT-OCOCF3, were still hidden. To better understand the disappearance of viable cells in the TBT-treated samples, we investigated the effects of TBT-OCOCF3, the compound that exhibited the milder cytotoxic activity, on the death of CAL-27 and MCF-10A cells using an annexin-V (Anx)/7-AAD double-staining assay. This assay allows us to simultaneously recognize viable cells (Anx-/7-AAD-), presumably early apoptotic cells (Anx+/7-AAD-), late apoptotic/secondary necrotic cells (Anx+/7-AAD+), and primary necrotic/lacking plasma-membrane-integrity cells (Anx-/7-AAD+). The cells were treated with 20 µM TBT-OCOCF3 (i.e., at a concentration showing appreciable toxicity), and, after 4 h (i.e., at a time Collectively, these data show that the organotin-derived compounds induce an immediate glucose uptake inhibition related to their cytotoxicity.

Effects of TBT-OCOCF 3 on Cell Death in CAL-27 and MCF-10A Cells
Data on the inhibition of the metabolic activity and the viable/dead cell count indicate that the CAL-27 cells were resistant to the cytotoxic effects exerted by cisplatin but quite susceptible to those induced by organotin derivatives, including TBT-OCOCF 3 . However, except for a certain relationship between toxicity and glucose uptake inhibition, the mechanisms involved in the cytotoxic effects of the organotin derivatives, particularly TBT-OCOCF 3 , were still hidden. To better understand the disappearance of viable cells in the TBT-treated samples, we investigated the effects of TBT-OCOCF 3 , the compound that exhibited the milder cytotoxic activity, on the death of CAL-27 and MCF-10A cells using an annexin-V (Anx)/7-AAD double-staining assay. This assay allows us to simultaneously recognize viable cells (Anx-/7-AAD-), presumably early apoptotic cells (Anx+/7-AAD-), late apoptotic/secondary necrotic cells (Anx+/7-AAD+), and primary necrotic/lacking plasma-membrane-integrity cells (Anx-/7-AAD+). The cells were treated with 20 µM TBT-OCOCF 3 (i.e., at a concentration showing appreciable toxicity), and, after 4 h (i.e., at a time when most of the cells were still detectable), the Anx+ and 7-AAD+ positivity was assessed using flow cytometer analysis. Moreover, to further investigate the mechanisms involved in TBT-OCOF 3 -induced cell death, attention was focused on emerging evidence that a complex crosstalk between apoptotic RCD and autophagy could play an essential role in influencing the survival/death of malignant cells and, consequently, the success of the chemotherapeutic treatment of cancer [30]. Therefore, tumorigenic CAL-27 cells were also subjected to cotreatment with the autophagy inhibitor wortmannin at 0.5 µM, in addition to TBT-OCOF 3 . Table 4 shows that TBT-OCOCF 3 induced approximately 4% Anx+/7-AADand 8% Anx-/7-AAD+ cells, compared with approximately 11% and 0.6%, respectively, in the control cells. A low but detectable amount of Anx+/7-AAD+ double-positive cells were induced by the treatment of TBT-OCOCF 3 , while this subset, as well as the Anx-/7-AAD+ subset, was practically absent in the cells treated with diluent alone as a control. Regarding MCF-10A cells, Table 4 shows that, after TBT-OCOCF 3 treatment for 4 h, the percentage of Anx+/7-AAD-cells significantly increased to 24.68% compared to 3.68% in the control cells, while that of the Anx-/7-AAD-cells significantly decreased from 75.96 to 14.16 compared to the control cells. Nevertheless, Anx+/7-AAD+ double-positive cells significantly increased at a higher percentage of 47.59 after treatment with respect to the 9.36% in the control cells. Therefore, TBT-OCOCF 3 treatment after 4 h induced both single Anx+ and, at the same time, double Anx+/7-AAD+ cells. Conversely, the percentage of Anx-/7-AAD+ was similar in the treated and control cells. This is coherent with the significant increase in the dead cell count by the trypan blue assay following 24 h of TBT-OCOCF 3 treatment, as shown in Figure 2b.  Regarding the effects of the autophagy inhibitor wortmannin on TBT-OCOCF 3 -induced cell death, Table 4 shows that the cotreatment did not significantly modify the percentage of Anx+/7-AAD-, while it increased the percentage of Anx+/7-AAD+ cells significantly, and that of the Anx-/7-AAD+ cells highly significantly, to approximately 6% and 29%, respectively. In addition, a cell cycle analysis carried out in the MCF-10A cells by PI staining revealed that a significant percentage of the TBT-OCOCF 3 -treated cells shifted from phase G2 (24%) to phase sub-G1 at 24 h after treatment, showing a significant increase in hypodiploid nuclei (15.4%), which are a typical feature of apoptotic cells, in comparison with the control cells ( Figure S4 in the Supplementary Materials).
These data suggest that TBT-OCOCF 3 -induced cell death resembling early and/or late apoptotic RCD, which might mainly be detectable in MCF-10 cells, after 4 h of treatment at the toxic concentration. However, at the same time, TBT-OCOCF 3 also induced a proneness to plasma membrane disruption, a typical feature of primary or secondary necrosis and other forms of RCD, both in the tumorigenic and the nontumorigenic cells used, as shown by the 7AAD+ positivity. In particular, this proneness was more pronounced in the MCF-10 cells in comparison with CAL-27 cells. Moreover, data on the effects of wortmannin were suggestive of a possible role of autophagy as a regulator of TBT-OCOCF 3 -induced cell death.

Effect of TBT-OCOCF 3 on Cell Death and Autophagy in U937 Cells
Based on the results of the cotreatment with TBT-OCOCF3 and wortmannin in CAL-27 cells, we decided to further investigate whether a crosstalk between apoptotic RCD and autophagy was effective in controlling cytotoxicity induced by organotins. To this purpose, nonadherent U937 cells, which are highly susceptible to TBT-induced cytotoxicity (Table 3; Figure S2 in the Supplementary Materials), were used. The cells were treated with 1 µM TBT-OCOCF 3 alone or with 0.5 µM wortmannin for 6 and 18 h. The percentages of cells showing morphological features of apoptotic cells were then evaluated by a microscopic analysis following staining with the fluorescent DNA-binding dye Hoechst. The choice for time and TBT-OCOCF 3 concentration was based on the previous results and those of preliminary experiments, showing that, in the selected experimental conditions but not at higher concentrations of TBT-OCOCF 3 , appreciable amounts of intact cells were still recoverable. In this round of experiments, fluorescence microscopic analysis indicated that, after 6 h, 15% of the U937 cells treated with TBT-OCOCF 3 alone showed morphological characteristics of apoptosis, while the addition of wortmannin significantly increased the portion of cells positive for apoptotic signs, overall up to nearly 37% (Figure 5a). A similar trend was observed when the timing was prolonged to 18 h but with much higher percentages of apoptotic cells (Figure 5a). As shown by representative images, the nuclear morphology of the U937 cells detected by Hoechst labeling revealed that the cotreatment of wortmannin even with a low dose of TBT-OCOCF 3 determined a considerable induction of cells with apoptotic features (Figure 5b).

Effect of TBT-OCOCF3 on Cell Death and Autophagy in U937 Cells
Based on the results of the cotreatment with TBT-OCOCF3 and wortmannin in C 27 cells, we decided to further investigate whether a crosstalk between apoptotic RCD autophagy was effective in controlling cytotoxicity induced by organotins. To this pose, nonadherent U937 cells, which are highly susceptible to TBT-induced cytotox ( Table 3; Figure S2 in the Supplementary Materials), were used. The cells were tre with 1 µM TBT-OCOCF3 alone or with 0.5 µM wortmannin for 6 and 18 h. The percen of cells showing morphological features of apoptotic cells were then evaluated by croscopic analysis following staining with the fluorescent DNA-binding dye Hoechst choice for time and TBT-OCOCF3 concentration was based on the previous results those of preliminary experiments, showing that, in the selected experimental condi but not at higher concentrations of TBT-OCOCF3, appreciable amounts of intact cells still recoverable. In this round of experiments, fluorescence microscopic analysis indic that, after 6 h, 15% of the U937 cells treated with TBT-OCOCF3 alone showed morph ical characteristics of apoptosis, while the addition of wortmannin significantly incre the portion of cells positive for apoptotic signs, overall up to nearly 37% ( Figure 5 similar trend was observed when the timing was prolonged to 18 h but with much h percentages of apoptotic cells (Figure 5a). As shown by representative images, the nu morphology of the U937 cells detected by Hoechst labeling revealed that the cotreat of wortmannin even with a low dose of TBT-OCOCF3 determined a considerable in tion of cells with apoptotic features (Figure 5b). To obtain direct information on the role of autophagy in regulating cytotoxicit duced by TBT-OCOCF3, the autophagic flux in viable cells was then assessed using a that selectively labels autophagic vacuoles. U937 cells were treated with 1 µM OCOCF3 alone and with the same amount of TBT-OCOCF3 plus 0.5 µM wortmannin To obtain direct information on the role of autophagy in regulating cytotoxicity induced by TBT-OCOCF 3 , the autophagic flux in viable cells was then assessed using a dye that selectively labels autophagic vacuoles. U937 cells were treated with 1 µM TBT-OCOCF 3 alone and with the same amount of TBT-OCOCF 3 plus 0.5 µM wortmannin for 6 and 18 h. These experimental conditions were the same, shown in Figure 5, in which increased levels of apoptosis following cotreatment with wortmannin were clearly detectable. As a positive control of autophagy induction, cells were treated for 18 h with 0.5 µM rapamycin alone or with the addition of the inhibitor wortmannin. All samples were analyzed for fluorescence emission using a fluorescence microplate reader at the indicated times. As illustrated in Figure 6a, only the rapamycin treatment determined a significant increase in autophagicspecific fluorescent green signals compared to the control cells. As expected, cotreatment with wortmannin significantly inhibited rapamycin-induced autophagy. Moreover, no significant change in fluorescence signals was found in the cells treated with TBT-OCOCF 3 as a single treatment or in combination with wortmannin after 6 or 18 h of treatment. Similar results were obtained when aliquots of the samples were analyzed by fluorescence microscopy. As shown in Figure 6b, autophagy, evidenced by green fluorescence, was present only in U937 cells treated with rapamycin.
Molecules 2023, 28, x FOR PEER REVIEW 12 of 21 expected, cotreatment with wortmannin significantly inhibited rapamycin-induced autophagy. Moreover, no significant change in fluorescence signals was found in the cells treated with TBT-OCOCF3 as a single treatment or in combination with wortmannin after 6 or 18 h of treatment. Similar results were obtained when aliquots of the samples were analyzed by fluorescence microscopy. As shown in Figure 6b, autophagy, evidenced by green fluorescence, was present only in U937 cells treated with rapamycin. Moreover, also in this set of experiments, the nuclear morphology of the U937 cells was detected by double staining with DAPI fluorescent dye. Again, the results exhibited that a cotreatment of TBT-OCOCF3 and wortmannin, at the concentration utilized to detect autophagy, determined a clear increase in cells with apoptotic features with respect to the samples treated with the TBT alone (Figure 6b, white arrows). These results confirmed what was previously observed using Hoechst as a DNA-binding dye to detect apoptosis, as shown in Figure 5. Moreover, also in this set of experiments, the nuclear morphology of the U937 cells was detected by double staining with DAPI fluorescent dye. Again, the results exhibited that a cotreatment of TBT-OCOCF 3 and wortmannin, at the concentration utilized to detect autophagy, determined a clear increase in cells with apoptotic features with respect to the samples treated with the TBT alone (Figure 6b, white arrows). These results confirmed what was previously observed using Hoechst as a DNA-binding dye to detect apoptosis, as shown in Figure 5.
These findings highlighted that wortmannin was able to noticeably increase the level of apoptotic RCD induced by a low dose of TBT-OCOCF 3 early after treatment. However, this was not owing to the inhibition of an autophagic flux that was not observed in cells treated with the compound in the same experimental conditions.

Discussion
The present in vitro comparative study shows that tributyltin derivatives were able to differently but always strongly affect cell proliferation, cell viability, and cell death in a tumorigenic, as well as in a nontumorigenic, cell line.
The biological activity of organotin(IV) compounds should depend on different features, such as the nature of the organic moiety and donor ligands attached to the tin atoms. Therefore, in this study, the major chemical physical parameters of the studied compounds were first ascertained. In particular, the solubility of the compounds in hydrophilic environments was checked through NMR before analyzing their biological effects. The NMR data show that a broad dose-effect range lower than the threshold of the solubility in phosphate buffer could be effectively utilized in biological assays with the tributyltin compounds. In any case, TBT compounds were dissolved in DMSO to make stock solutions for storage at concentrations high enough to warrant dilutions of at least 1/1000 with phosphate buffer before their use for in vitro and in vivo assays. Moreover, the results indicate that, in general, except for TBT-Cl, both TBT-OCOCF 3 and TBT-O did not remain aggregated at the concentrations utilized for the biological assays.
Regarding the biological activity, to obtain information on the possible anticancer potential of the tributyltin derivatives, as a first approach, the effects of the compounds on the cellular metabolic activity and on the cell viability of a tumorigenic and a nontumorigenic cell line were investigated. The effects towards PBMCs from healthy individuals and towards the nonadherent U937 cell line were also investigated for comparison. The results of this part of the study undoubtedly reveal the high cytotoxic properties of the tributyltin compounds. Among the three tested tributyltins, TBT-Cl was found to be significantly and equally highly inhibitory for the metabolic activity (IC 50 of approximately 1 µM) and cytotoxic both at high and low concentrations versus all tested cell lines. This might be owing to the electronegative characteristic of the chloride ion, which enhances the reactivity of the metal by attracting the electron cloud. Conversely, TBT-OCOCF 3 and TBT-O inhibited the metabolic activity similarly in all cell lines at higher values between 2 µM and 4 µM. In particular, TBT-OCOCF 3 (i.e., the synthesized tributyltin whose chemical and biological features were defined for the first time in this study) reduced the number of intact cells recovered after treatment in a dose-effect fashion and, to a lesser extent, in the MCF-10A cells with respect to the CAL-27 cells. Nevertheless, all cell lines assayed were more resistant to cisplatin treatment in comparison with the tributyltin derivatives. Although these results provide a clear picture of the high cytotoxic potential of tributyltins, they did not, however, provide sufficient information on events triggered by the compounds in the cells underpinning this cytotoxic activity. The substantial decrease in the metabolic activity, as assessed by the MTT assay, corresponds to an evident decrease in the number of cells in an active phase of their life. Nevertheless, this could be due to a metabolic block, to block in their cell cycle, to the entering of a high number of cells on the road towards one of the forms of RCD, or simply to the disappearance of intact cells in the treated samples due to the fact of their bursting because of primary or secondary necrosis. Even the trypan blue assays, despite demonstrating that the lowering of the metabolic activity was strictly associated with the lowering of the number of cells showing plasma membrane integrity, were helpful in elucidating this aspect. Unexpectedly, in our assays, the decrease in trypan-blue-negative, intact, and presumably viable cells was not balanced by a concomitant corresponding increase in trypan-blue-positive, intact, and presumably dead cells. On the other hand, viable trypan-blue-negative cells could not be distinguished from early apoptotic cells that they too were trypan blue negative, nor were the cells undergoing forms of RCD different from apoptosis with a loss of plasma membrane integrity distinguishable from primary or secondary necrotic cells, which were all trypan-blue-positive.
Thus, based on the results of the first part of the study, the successive phases mainly focused on the novel TBT-OCOCF 3 derivative, on its potentiality as a prototype anticancer candidate, on the attempt to characterize its cytotoxicity precisely, and, more specifically, on the type of induced cell death. However, a complex scenario was also found as a result of this part of the study. Experiments performed in the tumorigenic and nontumorigenic cells under study showed that a quite evident percentage of dead cells following TBT-OCOCF 3 treatment could be detectable only at higher concentrations of approximately 20 µM. In these experimental conditions, however, only a very low amount of total still-intact cells could be recovered after 24 h from the treated samples. Nevertheless, to understand which form of cell death could be induced by TBT-OCOCF 3 at the concentration mentioned above, cells with features of early and late apoptotic RCD and of necrotic death were distinguished by flow cytometry analysis following annexin-V/7AAD double staining early after treatment. The results show that a noticeable percentage of the MCF-10A cells that were recovered after 4 h of treatment with TBT-OCOCF 3 at 20 µM had the characteristics of early apoptosis. Nevertheless, a high percentage of cells were positive for both annexin-V and 7-AAD or for 7-AAD alone, indicating a coexistence of cells resembling early apoptotic, late apoptotic, and primary and secondary necrotic cells. Interestingly, the complexity of cell death induced by TBT was observed in a previous study demonstrating that at least two independent pathways were implicated in caspase-3-independent neuronal cell death caused by this compound [31].
Even though our results add an expected complexity to the phenomena under investigation (i.e., the actual fate of cells exposed to the selected tributyltin), they helped to address our efforts towards understanding, at least in part, the mechanisms that could control this network of cell-death-regulating signaling. Some key points concerning this aspect were defined. For example, our results demonstrate that cytotoxicity induced by the assayed triorganotin derivatives was found not to be owing to DNA binding. Indeed, unlike cisplatin, TBT-OCOCF 3 does not bind DNA, as demonstrated by the NMR analysis. This finding opens the way for developing TBT-OCOCF 3 -based compounds as potential anticancer agents in cisplatin-resistant tumors. Moreover, the cell cycle analysis showed that the cytotoxicity induced by TBT-OCOCF 3 was associated with a block in the G0/G1 and G2 phases and to the entry in the sub-G1 phase for the few intact cells that could be recovered after the treatment. Conversely, a very low percentage of cells were in the S phase. Similar findings were reported by other authors, who demonstrated that halogenated tin phosphinoyldithioformate complex-derived compounds induced the inhibition of proliferation but not accumulation in the S phase of the cell cycle [32]. Furthermore, the anticancer activity of di-and triorganotin(IV) compounds associated with D-(+)-galacturonic acid was reported to be associated with a block in the G0/G1 phase [20]. Therefore, our data and those reported by other authors lead us to conclude that even cells that can overcome the cytotoxicity after exposure to organotin compounds cannot proliferate at all, thus explaining the extremely low number of cells recovered by us following treatment and, most importantly, supporting their possible development as anticancer agents.
Another point of our study is the finding of a certain relationship between the cytotoxicity and the inhibition of the glucose metabolism of tin derivatives. We showed that TBT-OCOCF 3 is more effective in inhibiting glucose uptake in tumorigenic than in nontumorigenic cell lines. Glucose is a primary font of energy, and a hypothetical association between glucose metabolism and the effects of triorganotin derivatives was demonstrated in the human pluripotent embryonic carcinoma cell line NT2/D1 in which exposure to tributyltin inhibited glucose-6-phosphate and fructose-6-phosphate production via the inhi-bition of the transporter GLUT-1 [15]. In our model, CAL-27 exhibited a higher expression of membrane GLUT-1 with respect to MCF-10A cells (data not shown). Given that the dysregulation of the energy metabolism is a fundamental hallmark of cancer cells [33] and that glycolysis-related cancer cell survival is mediated by glucose transporters upregulated in some types of cancer [34], our results could suggest that compounds similar to TBT-OCOCF 3 could selectively act as cell-death inducers in tumors but not in normal cells. Nevertheless, it should be noted that TBT-OCOCF 3 markedly decreased the number of viable cells at concentrations that did not significantly affect glucose uptake. Thus, the role of the inhibition of glucose metabolism in the cytotoxicity exerted by tin compounds has yet to be elucidated.
The other key point addressed in our study is that a crosstalk between autophagy and cell death could be one intricate cell regulatory process triggered by tributyltins. The role of autophagy in controlling the fate of cancer cells is still a debated topic [30,35]. Autophagy is a double-edged sword whose activation/inhibition could favor tumor exhaustion [36]. Due to the evidence indicating a strict interaction between cell signaling controlling autophagy and different forms of cell death, including RCD and necrosis [37][38][39], targeting autophagy has been proposed as a new, possible strategy for anticancer therapy [40,41]. To better define events controlling tributyltin-induced cytotoxicity in CAL-27 cells-in fact, to address the possible regulatory role of autophagy-we utilized a cotreatment with an autophagy inhibitor, wortmannin. The results of our experiments in CAL-27 cells showed a low percentage of annexin-V+/7AAD-positive cells following treatment with TBT-OCOCF 3 alone, as expected, but the cotreatment with wortmannin led to the detection of an increased number of cells showing features of dead cells, including approximately 29% of annexin-V-/7AAD+ cells. This indicates a proneness to undergoing various forms of cell death, particularly necrosis, in tumorigenic cells in which signaling driving the autophagic flux was inhibited. Of fundamental importance, in this respect, for the continuation of the study was the utilization of nonadherent U937 cells as an experimental model, being more versatile for such an investigation. Even in the U937 cells, cotreatment with wortmannin significantly increased the percentage of dead cells treated with TBT-OCOCF 3 with respect to those treated with TBT-OCOCF 3 alone. In this case, it was possible to define that the increase in the number of dead cells should mainly be ascribed to cells resembling apoptotic cells. Interestingly, a shift of U937 towards a functional phenotype more susceptible to cell death due the fact of wortmannin cotreatment was observed even with low concentrations of TBT-OCOCF 3 and/or a short incubation time. However, the hypothesis that the levels of tributyltin-induced signaling driving towards apoptotic RCD should be negatively controlled by a concomitant triggering of autophagy was not confirmed by experiments finalized to prove this hypothesis directly. In the U937 cells, a low concentration of TBT-OCOCF 3 plus wortmannin induced twice the percentage of cells showing apoptotic features with respect to TBT-OCOCF 3 alone, but no sign of autophagic flux induction was observed as a consequence of tributyltin treatment. Wortmannin has been shown to inhibit autophagy through its well-known ability to suppress the class III phosphoinositide 3-kinase (PI3K), thus blocking the phosphorylation of phosphatidyl inositol (PI) that generates phosphatidylinositol 3-phosphate (PI3P), whose production is essential for the initiation of autophagy [42,43]. However, in our model, the inhibitory effect of wortmannin on autophagy seems not to be involved in the increase in the cytotoxic response. Thus, the effect of wortmannin in counteracting resistance to undergoing death in tumor cells might still be owing to its specific role of inhibiting the PI3K/AKT pathway. This pathway has been indicated as a central cellular mechanism for the phosphorylation of factors involved in the survival and migration of tumor cells [44]. In addition, the PI3/AKT kinase mammalian target of the rapamycin (mTOR) pathway is altered in HNSCC tumors, and agents targeting it are in clinical development to be used in combination treatment with chemotherapy [45]. Moreover, it has been recently reported that the conjugation of tributyltin with natural phenolic phytochemicals, such as ferulic acid (i.e., a ligand moiety absent within the TBT-OCOCF 3 structure), induced an increase in LC3II and p62 autophagic proteins that preceded cell death in colon cancer cells but neither apoptotic nor necroptotic cell death [21]. Interestingly, tumorigenic CAL-27 cells seem to undergo cell death through necrosis rather than apoptotic RCD following tributyltin treatment. In any case, our study, although it does not explain the effect of wortmannin, clearly indicates that a pharmacological intervention acting on the PI3K/AKT signaling pathway could dramatically potentiate the cytotoxic potential of TBT. Importantly, such a combination treatment seems able to overcome the resistance of tumor cells to the induction of death by tributyltins and to drive the fate of the treated cells towards a more specific and effective route of RCD.
This study established for the first time that the mechanisms underlying the induction of cell death by tributyltin derivatives seem unexpectedly very multifaceted. Based on our results, the induction of RCD or necrotic-related processes by TBT seems to not be a univocal phenomenon but rather an occurrence greatly dependent on the target cells, on the specific scaffold of the structure of the chemical derivatives, and on the concomitant modulation of specific cellular signaling pathways. Future studies are necessary to elucidate further the exact mechanisms underlying the cytotoxic effect of triorganotin derivatives and to promote the development of novel tributyltin compounds as anticancer agents.

Synthesis of Tributyltin Trifluoroacetate, Chemicals, and Reagents
The synthesis of tributyltin trifluoroacetate (TBT-OCOCF 3 ) was performed by the dropwise addition of TFA (0.04 mL, 0.5 mmol) to (Bu 3 Sn) 2 O (0.13 mL, 0.25 mmol). The reaction mixture was stirred at room temperature for 25 min and evaporated under reduced pressure to obtain a white solid. The compound was recrystallized from hexane and characterized by 1 H NMR, 13  TBT-O, TBT-Cl, and cisplatin were purchased from Sigma-Aldrich (San Diego, CA, USA). All tributyltin compounds were diluted in DMSO and stored at 1 M before their utilization for the biological assays. Wortmannin (Sigma-Aldrich) was diluted in DMSO and stored at 100 mM.
In all experiments carried out with the TBT compounds, the control cells were exposed, for the same amount of time as the treated cells, to control diluent alone corresponding to the higher concentration of the compounds assayed.

Cells
The high-tumorigenic human head and neck squamous cell carcinoma (HNSCC), adherent CAL-27 cells, and nonadherent U937 lymphoblastoid cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 U/mL streptomycin, 50 U/mL penicillin, and 2 mM glutamine (CM; Gibco-Invitrogen, Paisley, Scotland, United Kingdom) in a humidified incubator at 37 • C and 5% CO 2 . The nontumorigenic human mammary epithelial MCF-10A cells (ATCC, NIH, MD) were kept in DMEM-F12, (Lonza, Switzerland) supplemented with 5% horse serum (Invitrogen, Thermo-Scientific, CA, USA), 0.5 µg/mL hydrocortisone, 50 ng/mL cholera toxin, 0.01 mg/mL human insulin (Sigma-Aldrich), 50 U penicillin/streptomycin, and 2 µg/mL epidermal growth factor (EGF, Tebu-Bio, Le-Perray-en-Yvelines, France). The PBMCs were isolated from the buffy coat collected from healthy adult donor volunteers, who were seronegative for HIV and hepatitis B and C viruses, enrolled in the Polyclinic Hospital Tor Vergata Transfusion Center for blood donation for therapeutic purpose. The donors authorized the use of the remaining leukocytes for research purposes, signing a consent form. Anonymized buffy coats were diluted in phosphate buffered saline at pH 7 (PBS), and mononuclear cells were separated using a Ficoll-Hypaque density gradient (Cederlane, Hornby, Ontario, Canada) at a ratio of cells:gradient of 1:2. The cells were then centrifuged for 30 min at 1800 RPM and washed twice in RPMI 1640 medium (Gibco-Invitrogen). The PBMCs were stimulated with IL-2 at 10 U/mL (Proleukin, Chiron, Amsterdam, the Netherlands) before treatment with the compounds under study.

Metabolic Activity and Viability Assay
The inhibition of cell metabolic activity, revealed by the reduction of the oxidative burst, was performed through a colorimetric method based on the reduction of tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich), with the formation of formazan crystals solubilized following the addition of SDS lysis buffer. The MTT reagent was diluted in sterile PBS (phosphate buffered saline) and stored at a concentration of 5 mg/mL, while 20 g of SDS was diluted in 100 mL solution composed of 50 mL bidistilled water and 50 mL dimethylformamide (Sigma-Aldrich). The assay was performed by seeding 5 × 10 3 cells in 100 µL into a 96-well plate in the presence or absence of different concentrations of organotin compounds and the reference drug. After 18 h of incubation at 37 • C, 10 µL of MTT was added, and, after 6 h, 100 µL of SDS was added. After overnight incubation, the optical density was read by a spectrophotometer (Packard, Spectral Count Microplate Photometer) at the wavelength of 570 nanometers. The amount of formazan produced was directly proportional to the number of alive cells. The results are expressed as the drug concentration required to inhibit 50% of the metabolic activity ± standard deviation (IC 50 ± SD). The concentrations of the compounds to be tested and the length of the incubation time for the MTT assay were chosen based on preliminary experiments showing IC 50 values in the low micromolar range for TBT at 24 h and very small amounts of cells still detectable after longer incubation times. A viability assay was performed using the trypan blue exclusion test, with the concentrations of the compounds and timing selected on the basis of the MTT assay and preliminary trypan blue exclusion tests.

NMR Spectroscopy
For the NMR studies, the compounds were dissolved in DMSO-d6 and a phosphate buffer (50 mM, 5% D 2 O, pH = 7.4) containing, as an internal standard, 2,2,4,4tetradeuterumtrimethylsilylpropionic acid (TSP). The NMR experiments were performed in D 2 O at 25 • C and recorded with a Bruker Avance spectrometer operating at 700 MHz for 1 H, equipped with a 5 mm inverse TXI probe, z-axis gradients, and a Sample Xpress Lite autosampler. The 1 H-NMR spectra were recorded with a spectral window of 15 ppm, 16 k complex points, and a relaxation delay of 10 s for a total of 16 transients. The 1 H-1 H COSY experiments were acquired with spectral windows of 13 × 11 ppm (carrier frequency of 5 ppm) using 2048 × 128 data points, 2 transients, and a relaxation delay of 2 s. The 1 H-13 C HSQC experiments were acquired with spectral windows of 13 × 70 ppm (carrier frequencies of 5 and 30 ppm) using 4096 × 56 data points, 4 transients, and a relaxation delay of 2 s. The 1 H-1 H TOCSY experiments, implemented with an excitation sculpting scheme for the water suppression [46], were conducted with spectral windows of 20 × 19 ppm (carrier frequency of 4.7 ppm) using 4096 × 256 data points, 24 transients, relaxation delay of 2 s, and a mixing time of 80 ms.
The solubility was determined by preparing a 600 µL solution for each compound in DMSO and buffer solution with a nominal concentration of 400 µM. The effective concentrations were measured at 37 • C using 3-(trimethylsilyl) propionic 2,2,3,3-d 4 acid (TSP) as an internal standard.

Glucose Uptake
To evaluate the glucose uptake, both the CAL-27 and MCF-10 A cell lines were seeded at 2 × 10 5 in 24 plates and incubated overnight at 37 • C. After 24 h, the cells were washed in warm PBS at pH 7.4 and diluted in media without glucose for 40 min. The cells were then washed in PBS and resuspended in suitable complete media and treated with different concentrations of the compounds to assay and incubated with a fluorescent glucose analog (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino] D-glucose) (2-NBDG) (Sigma-Aldrich) for 1 h at 37 • C. At the end of incubation, the cells were detached through a trypsin solution and PBS-EDTA (without Ca2+ and Mg), washed in PBS, resuspended in FACS flow, and analyzed with flow cytometry through the software Cell Quest (BD Bioscience).

Cell Death and Autophagy
Early RCD-related events were detected through double staining of the cells with fluorescent annexin-V, which preferentially binds phosphatidylserine that appears very early in apoptosis at the external cell surface, and with 7-amino actinomycin D (7-AAD) solution, as a viability dye. The "Annexin V-FITC Kit 7-AAD KiT" (IM3614, Beckman Coulter) was used according to the manufacturer's instructions. Briefly, 5 × 10 5 cells were incubated for 15 min with annexin-V-fluorescein isothiocyanate and washed in annexin buffer. Cells were then stained with 7-AAD and analyzed immediately after staining by flow cytometry analysis. The data acquisition and analyses were performed using CytExpert 2.0 (Beckman Coulter, United States) on at least 150,000 events for each sample.
Apoptotic RCD was evaluated in U937 cells by morphological analysis following staining with Hoechst chromatin dye, as previously described [47].
Autophagy was evaluated in the U937 cells using the Autophagy Assay kit from Abcam (ab139484, Abcam, Cambridge, UK) according to the manufacturer's protocol. In particular, the kit can measure autophagic vacuoles and monitor autophagic flux in live cells using an optimized dye that selectively labels autophagic vacuoles. The 488 nm excitable fluorescent green detection reagent (i.e., green dye) supplied in the kit becomes brightly fluorescent in vesicles produced during autophagy. The nuclear counterstain DAPI (i.e., nuclear dye) is provided in the kit as well to highlight cellular nuclei. The cells were seeded in 24-well plates at a density of 0.5 × 10 6 cells/mL and treated with vehicle, wortmannin (Sigma-Aldrich W1628), TBT-OCOCF 3 , or a combination of the two for 6 and 18 h. As a positive control, cells were treated for 18 h with the autophagy-inducer rapamycin (Sigma-Aldrich R0395) alone or in combination with the inhibitor wortmannin. At the end of the treatment, samples were collected, washed with 1X assay buffer, and incubated with 100 µL of a dual-detection reagent (1X fluorescent green reagent plus 1X nuclear dye in assay buffer) for 30 min at 37 • C in the dark. Next, the cells were carefully washed three times, and the fluorescence intensity was measured using an appropriate filter set for a fluorescent green reagent (excitation: 463 nm/emission: 534 nm) and nuclear dye (excitation: 350 nm/emission: 461 nm) in a Fluoroskan microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The autophagic green fluorescence intensity was expressed as the fold change with respect to the control cells using values of the relative fluorescence units (RFUs) measured for green fluorescence normalized to the RFUs measured for the nuclear dye fluorescence in the same sample to control for any change in the number of cells in the samples subjected to different treatments. After washing, aliquots of stained cells were also analyzed by fluorescence microscopy (Leica Leitz DMRE, Wetzlar, Germany). For each sample, images from the same field were taken with a green (for green autophagyspecific reagent) or blue filter (for nuclear stain). Representative fields were photographed using 400× magnification.

Statistical Analysis
The data analysis was performed using the SPSS statistical software system (version 17.0 for Windows, Chicago, IL, USA). The data were assessed using parametric one-way analysis of variance (ANOVA). The statistical significance of the differences among groups were calculated using Bonferroni's post hoc multiple comparison methods. The results of the statistical tests are reported in the figures and tables.

Conclusions
In conclusion, the data reported in this study highlight how the final fate of cells exposed to tributyltin compounds is strongly affected by the balance of signals triggering the different forms of RCD and necrosis and of the PI3K/AKT pathway. The elucidation of these aspects could play a key role in the design and synthesis of organotin derivatives alone, and/or in conjugation with a suitable vehicle, or in combination with survival inhibitors to obtain clinically helpful cocktail drugs specifically active towards malignant cells.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28093856/s1; Figure S1: Regions of the 1 H-NMR spectra of TBT compounds in DMSO; Figure S2: Viability of U937 cells following treatment with TBT-OCOCF 3 ; Figure   Institutional Review Board Statement: Ethical review and approval were waived for this study because only anonymized buffy coats from healthy donor volunteers who signed a written informed consent form were utilized as a source of PBMCs, and the study was conducted in accordance with the Declaration of Helsinki.